Method for detecting enterocytozoon hepatopenaei through multienzyme isothermal amplification-colloidal gold

Abstract

The invention discloses a multienzyme isothermal amplification-colloidal gold detection method for enterocytozoon hepatopenaei, and relates to the technical field of pathogen detection, the method comprises the following specific steps: specific primer and probe design: based on an EHP specific PTP3 gene, designing a primer pair and a probe according to specific parameters, it is guaranteed that the primer concentration is appropriate, and probe labeling and modification meet the detection requirements; sample nucleic acid extraction: selecting specific tissues of the suspected EHP-infected prawns, extracting nucleic acid by adopting a kit according to a standard process, and detecting the quality of the nucleic acid; the specific PTP3 gene of the enterocytozoon hepatopenaei is taken as a target, so that the target pathogen can be accurately detected, the sensitivity of the MIRA-LFD system is high, the low-concentration pathogen in the initial stage of enterocytozoon hepatopenaei infection can be detected, missing detection of a recessive infection sample is effectively avoided, and the detection sensitivity is high. Reliable technical support is provided for prawn offspring seed quarantine, regular screening in the breeding process, diseased prawn group diagnosis and epidemiological monitoring, and early warning and precise prevention and control of epidemic situations can be achieved.

Description

Technical Field

[0001] This invention relates to the field of pathogen detection technology, specifically a method for detecting Enterocytozoa hepatis in shrimp using a multi-enzyme isothermal amplification-colloidal gold method. Background Technology

[0002] Enterocytozoon hepatocellular carcinoma (EHP) is a significant threat to shrimp farming. It primarily infects the hepatopancreatic ducts and epithelial cells of various farmed shrimp species, including Penaeus monodon, Litopenaeus vannamei, and Litopenaeus vannamei. This causes epithelial cell shedding and the formation of intracytoplasmic inclusion bodies. Although it does not directly lead to acute high mortality, it severely interferes with the digestion, absorption, and energy metabolism of shrimp, resulting in stunted growth, significant differences in body size, weakened immune function, and increased risk of secondary infections. Furthermore, EHP has complex transmission routes, including vertical transmission from parent shrimp to seedlings and horizontal transmission through ingestion of contaminated feces, damaged tissues, or spores stably present in the water. This can lead to rapid outbreaks within farms, causing substantial economic losses to the industry.

[0003] Currently, EHP detection methods mainly include traditional histopathological methods and molecular biological methods. Traditional histopathological methods, such as HE staining and Masson staining, require microscopic observation of spores or pathological changes, rely on 40-100x oil immersion and professional operating experience, and immature spores are difficult to identify. The operation is cumbersome and time-consuming, which cannot meet the needs of large-scale rapid screening. In situ hybridization (ISH) technology has high specificity, but the process is complex and costly, and it is also not suitable for large-scale screening. Among molecular biological methods, real-time quantitative PCR can accurately quantify, but it relies on sophisticated instruments and has high detection costs, which limits its application in aquaculture. Loop-mediated isothermal amplification (LAMP) technology does not require a thermal cycler, but it requires the design of 6 primers, and multiplex amplification is prone to non-specific pairing, resulting in a high risk of false positives. Conventional PCR has good specificity and sensitivity, but it relies on a thermal cycler and has a long detection cycle, which cannot meet the needs of immediate detection in aquaculture. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for detecting EHP-specific enterocytozoa in shrimp using multi-enzyme isothermal amplification-colloidal gold. This method utilizes EHP-specific PTP3 genes as targets, designed with dedicated primers and probes, to construct a detection system combining multi-enzyme isothermal rapid amplification (MIRA) with colloidal gold lateral flow chromatography (LFD) strips. The MIRA reaction, conducted at an isothermal temperature of 25-40℃, completes specific amplification within 20 minutes. The MIRA-LFD system visualizes results via the test strip, achieving a detection limit of 1.08 × 10¹ copies / μL, and exhibits no cross-reactivity with other common aquatic pathogens. Clinical sample detection results are completely consistent with PCR. This method offers advantages such as speed, sensitivity, and ease of operation, providing crucial technical support for early warning and control of EHP.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for detecting Enterocytozoa hepatis in shrimp using multi-enzyme isothermal amplification-colloidal gold, the method comprising the following specific steps:

[0006] Specific primer and probe design: Based on the EHP-specific PTP3 gene, primer pairs and probes are designed according to specific parameters, and the primer concentration is appropriate and the probe labeling and modification meet the detection requirements.

[0007] Nucleic acid extraction from samples: Specific tissues of shrimp suspected of being infected with EHP were selected, and nucleic acid was extracted using a kit according to standard procedures. The quality of the nucleic acid was then tested.

[0008] MIRA amplification reaction: Prepare the MIRA reaction system by mixing buffer, primers, enzyme mixture, and template nucleic acid components in the correct proportions, and react under suitable isothermal conditions to complete the efficient amplification of the target gene;

[0009] Colloidal gold LFD detection: Add the amplification product to the sample well of the colloidal gold test strip, let it stand at room temperature, and judge the test results according to the appearance of the bands in the detection area and the quality control area according to the standard rules.

[0010] Furthermore, in the specific primer and probe design step, primer pairs and probes are designed using PrimerPremier software. Primers are 25-30 bp in length with a GC content of 45%-55%, and probes are 30-35 bp in length. The 5' end is labeled with a FAM fluorescent group, a THF modification site is inserted in the middle, and the 3' end is labeled with a C3spacer quencher group. The primer concentration is set to 10 μmol / L. The primers include upstream primers F1-F7 and downstream primers R1-R7, and the probes include P1 and P2. The primer and probe sequences are as follows:

[0011] F1: 5'-CACCAGAAGAGGCACAGGCTTTGAAGAATAC-3';

[0012] F2:5’-CTTATCCTTTCCCACACGGTCACAGCCGTAC-3’;

[0013] F5:5’-GCACTGGATGAGTTAGTTCAGCACGCATATG-3’;

[0014] F6:5’-CAGCACGCATATGACACATCAATTGATATG-3’;

[0015] F7:5’-CAATTGATATGGGCATGACACCTGAAGAAG-3’;

[0016] R1:5’-Biotin-GATGAAGGAGAACCGAGTGGAGTATAGTCTTC-3’;

[0017] R2:5’-Biotin-GCTAAACTTTGGAGATGAAGGAGAACCGAG-3’;

[0018] R3:5’-Biotin-CTTCGTCCAGCGGTGGTGAAGTTGGGCTAAAC-3’;

[0019] R4:5’-Biotin-CATATCCTGACGTGCTTCGTCCAGCGGTGGTG-3’;

[0020] R5:5’-Biotin-GCACGATCATCTGCTTCTTGTGGTGAATAAC-3’;

[0021] R6:5’-Biotin-GCGTGAAACATCCTTAGCCTTGTTGTTAC-3’;

[0022] R7:5’-Biotin-CAAGTGAACCTGGTGTGATTTCCTCTGCTG-3’;

[0023] P1:[5’FAM]-CAACACAGCATGGTAGTCAATATGGTGATG[THF]GTGGTACACAGAAAC-[3’C3spacer];

[0024] P2:[5’FAM]-CTCAGAAGCAACCCGTTCATATGTTGAAAC[THF]TACCACGACTACCT-[3’C3spacer]。

[0025] Furthermore, in the sample nucleic acid extraction step, nucleic acid is extracted using a kit according to the standard procedure: 200 μL of lysis buffer and 20 μL of proteinase K are added to the tissue, and the mixture is incubated at 56°C for 30 minutes until the tissue is completely digested; then 200 μL of anhydrous ethanol is added, mixed by inversion, and transferred to the adsorption column, centrifuged at 12000 rpm for 1 minute, and the waste liquid is discarded; 500 μL of low-salt buffer is added, centrifuged at 12000 rpm for 1 minute, and the waste liquid is discarded; then 700 μL of ethanol buffer is added, centrifuged at 12000 rpm for 1 minute, the waste liquid is discarded, and this process is repeated once; then the empty column is centrifuged at 12000 rpm for 2 minutes to remove residual washing liquid; 50 μL of enzyme-free water is added to the center of the adsorption column, the column is allowed to stand at room temperature for 5 minutes, centrifuged at 12000 rpm for 2 minutes, and the eluent is collected.

[0026] Furthermore, in the sample nucleic acid extraction step, the nucleic acid concentration and purity are determined using a UV spectrophotometer, requiring a concentration of 10-100 ng / μL and an OD260 / OD280 ratio of 1.8-2.0.

[0027] Furthermore, in the MIRA amplification reaction step, a 25 μL MIRA reaction system is constructed, comprising: 12.5 μL of 2×MIRA buffer to provide the required pH environment and dNTPs; 1 μL of 10 μmol / L upstream primer to bind to the upstream sequence of the target gene; 1 μL of 10 μmol / L downstream primer to bind to the downstream sequence of the target gene; 0.5 μL of DNA helicase to unwind the double-stranded DNA of the target gene; 0.5 μL of recombinase to mediate the binding of the primer to the single strand of the target gene; 0.5 μL of SSB protein to stabilize the single-stranded DNA; 0.5 μL of DNA polymerase to extend the primer and synthesize a new DNA strand; 2 μL of template nucleic acid, i.e., the nucleic acid of the sample to be tested; and 5 μL of enzyme-free water to complete the system volume.

[0028] Furthermore, in the MIRA amplification reaction step, the prepared reaction system is gently mixed and placed in a constant temperature water bath at 25–42°C for 5–20 minutes.

[0029] Furthermore, in the colloidal gold LFD detection step, the colloidal gold lateral flow chromatography test strip consists of a sample pad, a colloidal gold conjugate pad, a nitrocellulose membrane, and an absorbent pad. The colloidal gold conjugate pad is coated with a monoclonal antibody-colloidal gold complex containing an anti-FAM fluorescent group; the detection area on the NC membrane is coated with anti-biotin antibody, and the control area is coated with goat anti-mouse IgG antibody.

[0030] Furthermore, in the colloidal gold LFD detection step, 10 μL of MIRA amplification product is taken and added to the sample pad of the test strip, and left to stand at 20-25℃ for 5-8 minutes until the liquid has completely migrated to the absorbent pad.

[0031] Furthermore, in the colloidal gold LFD detection step, a positive result is indicated by clear red bands appearing in both the detection area (T zone) and the control area (C zone), indicating that the sample contains the EHPPTP3 gene; a negative result is indicated by a red band appearing only in the control area (C zone), indicating that the sample does not contain the EHPPTP3 gene; an invalid result is indicated by no red band appearing in the control area (C zone), regardless of whether the detection area (T zone) shows color, indicating that the test strip is invalid and needs to be tested again.

[0032] Compared with existing technologies, this method for detecting Enterocytozoa hepatis in shrimp using a multi-enzyme isothermal amplification-colloidal gold method has the following advantages:

[0033] I. This invention uses the PTP3 gene, unique to Enterocytozoa hepatis, as a target to accurately detect the target pathogen. Moreover, the MIRA-LFD system has high sensitivity and can detect low concentrations of the pathogen in the early stages of Enterocytozoa hepatis infection, effectively avoiding missed detection of latent infection samples. It provides reliable technical support for shrimp seedling quarantine, regular screening during the farming process, diagnosis of diseased shrimp populations, and epidemiological monitoring, which helps to achieve early warning and precise control of epidemics.

[0034] II. This invention combines multi-enzyme isothermal amplification (MIRA) technology with colloidal gold side-flow chromatography (LFD) test strips, which greatly shortens the detection time and meets the needs of on-site detection in aquaculture. At the same time, this method does not require sophisticated equipment such as thermal cyclers and gel imaging systems. Amplification can be completed using only a constant temperature water bath. The results of the colloidal gold test strips are intuitive and easy to interpret. Farmers can operate the method after simple training, providing a convenient and effective detection method for grassroots aquaculture personnel.

[0035] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0037] Figure 1 A schematic diagram showing the amplification results of different MIRA-LFD primer-probe combinations;

[0038] Figure 2 Schematic diagrams illustrating the optimization of reaction conditions for the MIRA (a, c) and MIRA-LFD (b, d) methods;

[0039] Figure 3 A schematic diagram showing the specificity detection results of the MIRA(a) and MIRA-LFD(b) methods;

[0040] Figure 4 This is a schematic diagram showing the sensitivity detection results of PCR (a), MIRA (b), and MIRA-LFD (c) methods;

[0041] Figure 5 Schematic diagrams of clinical sample testing results using PCR (a) and MIRA-LFD (b, c, d) methods;

[0042] Figure 6 This is a flowchart of a method for detecting shrimp hepatic enterocytozoa using a multi-enzyme isothermal amplification-colloidal gold method. Detailed Implementation

[0043] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0044] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for detecting EHP-specific enterocytozoa in shrimp using multi-enzyme isothermal amplification-colloidal gold. This method utilizes EHP-specific PTP3 genes as targets, designed with dedicated primers and probes, to construct a detection system combining multi-enzyme isothermal rapid amplification (MIRA) with colloidal gold lateral flow chromatography (LFD) strips. The MIRA reaction, conducted at an isothermal temperature of 25-40℃, completes specific amplification within 20 minutes. The MIRA-LFD system visualizes results via the test strip, achieving a detection limit of 1.08 × 10¹ copies / μL, and exhibits no cross-reactivity with other common aquatic pathogens. Clinical sample detection results are completely consistent with PCR. This method offers advantages such as speed, sensitivity, and ease of operation, providing crucial technical support for early warning and control of EHP.

[0045] The following is a specific implementation method of the multi-enzyme isothermal amplification-colloidal gold detection method for shrimp hepatic enterocytozoa provided by the present invention:

[0046] Preparation of experimental materials

[0047] Reagents:

[0048] MIRA enzyme mixture (containing DNA helicase, recombinase, single-stranded DNA binding protein SSB, and DNA polymerase), 2×MIRA buffer;

[0049] 10 μmol / L shrimp enterocytozoon hepatocytozoon (EHP) PTP3 gene-specific primer pairs and probes: Forward primers: F1 (5'-CACCAGAAGAGGCACAGGCTTTGAAGAATAC-3'), F2 (5'-CTTATCCTTTCCCACACGGTCACAGCCGTAC-3'), F5 (5'-GCACTGGATGAGTTAGTTCAGCACGCATATG-3'); Reverse primers: R1 (5' end labeled with biotin, 5'-Biotin-GATGAAGGAGAACCGAGTGGAGTATAGTCTTC-3'), R3 (5' end labeled with biotin, 5'-Biotin-CTTCGTCCAGCGGTGGTGAAGTTGGG CTAAAC-3'), R5 (5' end labeled with Biotin, sequence 5'-Biotin-GCACGATCATCTGCTTCTTGTGGTGAATAAC-3'), probes P1 (5' end labeled with FAM, containing a THF modification site in the middle, 3' end labeled with C3spacer, sequence 5'FAM-CAACACAGCATGGTAGTCAATATGGTGATG[THF]GTGGTACACAGAAAC-[3'C3spacer]) and P2 (5' end labeled with FAM, containing a THF modification site in the middle, 3' end labeled with C3spacer, sequence 5'FAM-CTCAGAAGCAACCCGTTCATATGTTGAAAC[THF]TACCACGACTACCT-[3'C3spacer]).

[0050] DNA extraction kit (including lysis buffer, proteinase K, washing buffer I, washing buffer II, adsorption column, and enzyme-free water);

[0051] Colloidal gold lateral flow chromatography test strip (detection zone T area coated with anti-biotin antibody, control zone C area coated with goat anti-mouse IgG antibody, colloidal gold conjugate pad coated with anti-FAM fluorescent monoclonal antibody-colloidal gold complex).

[0052] EHP standard plasmid (concentration 1.08 × 10⁻⁶) 7 copies / μL, pre-construction in the laboratory), ddH2O (enzyme-free);

[0053] Routine PCR control reagents: 10 μmol / L PCR-514F primers (sequence 5'-TTGCAGAGTGTTGTTAAGGGTTT-3'), 10 μmol / L PCR-514R primers (sequence 5'-CACGATGTGTCTTTGCAATTTTC-3'), Taq DNA polymerase, 2× PCR buffer, and dNTP mixture.

[0054] Instruments: constant temperature water bath (accuracy ±0.5℃), ultraviolet spectrophotometer, high-speed centrifuge (maximum speed ≥12000rpm), gel imaging system, PCR instrument (for routine PCR control experiments), sterile centrifuge tubes (1.5mL), pipettes (10μL, 20μL, 100μL, 1000μL), DL2000 DNA Marker.

[0055] Samples: Eight Litopenaeus vannamei samples (numbered S1-S8), eight Penaeus monodon samples (numbered S9-S16), and eight Litopenaeus vannamei samples (numbered S17-S24) suspected of being infected with EHP were collected from a shrimp farm, totaling 24 clinical samples; at the same time, an EHP-negative shrimp sample (numbered N1) and an EHP-positive control sample (numbered P1, known to contain EHP genomic DNA) were also prepared.

[0056] Experimental steps:

[0057] Screening and preparation of specific primer-probe combinations:

[0058] refer to Figure 1 The amplification results of different MIRA-LFD primer-probe combinations are shown. The optimal combination with clear amplification bands and strong positive signals is selected first. In this example, three optimal combinations are selected: F1 / R1-P1, F2 / R3-P1, and F5 / R5-P2. The above primers and probes are diluted with enzyme-free water to a working concentration of 10 μmol / L, aliquoted and stored at -20℃. Avoid repeated freeze-thaw cycles. Equilibrate to 25℃ at room temperature before use.

[0059] Nucleic acid extraction from samples:

[0060] Sample processing: Take 0.15g of liver and pancreatic tissue from 24 clinical samples and N1 and P1 samples respectively, place them in sterile 1.5mL centrifuge tubes, cut them into small pieces with sterile scissors, add 200μL of lysis buffer from the commercial kit and 20μL of proteinase K, mix well and incubate in a 56℃ constant temperature water bath for 30 minutes, gently inverting the centrifuge tube once every 10 minutes until the tissue is completely digested (the solution after digestion is clear and free of flocculent precipitate).

[0061] Nucleic acid purification: Add 200 μL of anhydrous ethanol to the digested solution, quickly invert the centrifuge tube 10 times to mix thoroughly. At this time, a small amount of white flocculent precipitate can be observed in the solution. Transfer the above mixture to the adsorption column provided with the kit (the adsorption column is pre-placed on the collection tube), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0062] Washing to remove impurities: Add 500 μL of the washing buffer I provided with the kit to the adsorption column, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid; then add 700 μL of the washing buffer II provided with the kit to the adsorption column, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid. Repeat this washing step once (a total of 2 treatments with washing buffer II); put the adsorption column back into the collection tube, centrifuge the empty column at 12000 rpm for 2 minutes to completely remove the residual washing buffer in the adsorption column (to avoid ethanol residue affecting the subsequent MIRA amplification enzyme activity).

[0063] Nucleic acid elution: Transfer the adsorption column to a new sterile 1.5 mL centrifuge tube, add 50 μL of enzyme-free water to the center of the adsorption column membrane, and let stand at room temperature for 5 minutes to allow the nucleic acid to fully dissolve in the enzyme-free water; centrifuge at 12000 rpm for 2 minutes, and collect the elution buffer in the centrifuge tube, which is the total nucleic acid of each sample.

[0064] Nucleic acid quality testing: The concentration and purity of nucleic acid in each sample were determined using a UV spectrophotometer. The results showed that the concentration of nucleic acid in all samples was within the range of 35-60 ng / μL, and the OD260 / OD280 value was between 1.82 and 1.95, which met the quality requirements for MIRA amplification and routine PCR detection (concentration 10-100 ng / μL, OD260 / OD280 = 1.8-2.0).

[0065] MIRA amplification reaction:

[0066] Reaction system configuration: Refer to Figure 2 The optimized MIRA reaction conditions shown indicate that 40.0℃ and 20 minutes were selected as the optimal reaction parameters. Following a 25μL system specification, the following components were added sequentially to a sterile PCR tube: 12.5μL 2×MIRA buffer, 1μL 10μmol / L upstream primer, 1μL 10μmol / L downstream primer, 0.5μL DNA helicase, 0.5μL recombinase, 0.5μL SSB protein, 0.5μL DNA polymerase, 2μL sample nucleic acid, and 5μL enzyme-free water. Each sample was amplified using three primer / probe combinations: F1 / R1-P1, F2 / R3-P1, and F5 / R5-P2. A positive control (using P1 sample nucleic acid as template, 2μL) and a blank control (using enzyme-free water instead of template, 2μL) were also included. Each reaction was performed in triplicate.

[0067] Amplification reaction: Gently invert the prepared PCR tube to mix (avoid air bubbles to prevent affecting enzyme activity), place it in a 40.0℃ constant temperature water bath, and time the reaction for 20 minutes; simultaneously set up temperature gradient validation groups (36℃, 38℃, 42℃, 44℃, 46℃) and time gradient validation groups (5 minutes, 10 minutes, 15 minutes, 30 minutes), as per [reference needed]. Figure 2 Experimental designs a, b, c, and d were used to verify the amplification effect under different conditions. After the reaction was completed, the PCR tubes were removed and cooled to 25°C at room temperature for later use.

[0068] Colloidal gold LFD detection:

[0069] Detection procedure: Take 10 μL of each of the cooled MIRA amplification products and add them to the corresponding wells of the colloidal gold side-flow chromatography test strip. Place the test strip flat on a clean lab bench and let it stand at room temperature (22-25℃) for 5 minutes. Avoid touching or moving the test strip during this time to ensure that the liquid migrates to the absorbent pad at a uniform speed.

[0070] Result interpretation: Refer to Figure 1 , Figure 3 , Figure 4 , Figure 5 Result interpretation criteria: Observe the test strip bands after 5 minutes.

[0071] Positive control (P1 sample): All repeat test strips of the 3 sets of primer probe combinations showed clear red bands in both the detection area (T area) and the quality control area (C area), which was judged as EHP positive;

[0072] Blank control: Only the control area (C area) of all repeated test strips showed a red band, and the test area (T area) showed no color development, which was judged as EHP negative;

[0073] Negative control (N1 sample): All duplicate test strips showed a red band only in the control area (C area), which was judged as EHP negative;

[0074] Clinical samples: S2, S5, S7, S10, S13, S15, S18, and S22 (a total of 8 samples), all showed double red bands in both the T and C regions on the repeat test strips of the 3 primer-probe combinations, and were judged to be EHP positive; the remaining 16 samples (S1, S3, S4, S6, S8, S9, S11, S12, S14, S16, S17, S19, S20, S21, S23, and S24) showed only color in the C region, and were judged to be EHP negative;

[0075] Gradient verification results: Within the temperature range of 36-42℃, the T zone of the test strip showed the deepest color at 40℃; the T zone showed clear color at 15-20 minutes, while some samples showed no color in the T zone at 5 minutes and 10 minutes; there was no significant difference in color development between 30 minutes and 20 minutes, further verifying that 40℃ and 20 minutes are the optimal reaction conditions.

[0076] Routine PCR control test:

[0077] To verify the accuracy of the method of the present invention, refer to Figure 5 The experimental design for a involved routine PCR testing of 24 clinical samples and N1 and P1 samples:

[0078] PCR system preparation: 25 μL system contains 12.5 μL of 2×PCR buffer, 1 μL of 10 μmol / L PCR-514F primer, 1 μL of 10 μmol / L PCR-514R primer, 2 μL of dNTP mixture, 0.5 μL of Taq DNA polymerase, 2 μL of sample nucleic acid, and 5.5 μL of enzyme-free water.

[0079] PCR reaction conditions: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 30 seconds, for a total of 35 cycles; 72℃ final extension for 10 minutes, and storage at 4℃.

[0080] Results detection: 10 μL of PCR product was subjected to 1.5% agarose gel electrophoresis. The DL2000 DNA Marker was used as a reference, and the results were observed using a gel imaging system. The results showed that the 8 positive samples (S2, S5, S7, S10, S13, S15, S18, and S22) detected by conventional PCR were completely consistent with the detection results of the MIRA-LFD method of this invention, with a positive concordance rate and a negative concordance rate of 100%.

[0081] Specificity and sensitivity verification:

[0082] Specificity verification: Refer to Figure 3 The experimental design used genomic DNA of Decapoda iridovirus, infectious myonecrosis virus, leukoplakia syndrome virus, acute hepatopancreatic necrosis virus, vibrio parahaemolyticus, and Vibrio parahaemolyticus as interference templates. The method of this invention was used for detection. The results showed that only the EHP positive template (P1 sample) showed double bands in the T and C regions, while the other interference templates showed only color in the C region, proving that the method has no cross-reactivity and high specificity.

[0083] Sensitivity verification: Refer to Figure 4 The experimental design involved serially diluting the EHP standard plasmid to 1.08 × 10⁻⁶. 7 1.08×10 6 1.08×10 51.08×10 4 1.08×10³, 1.08×10², 1.08×10¹, 1.08×10 0 The results of MIRA-LFD assay showed that the method could detect a minimum of 1.08 × 10¹ copies / μL of standard plasmid, which is significantly more sensitive than conventional PCR (the minimum detection limit of conventional PCR is 1.08 × 10² copies / μL).

[0084] Experimental conclusion:

[0085] This embodiment, through testing 24 clinical samples, specificity verification, sensitivity verification, and conventional PCR control experiments, confirms that using the optimal primer and probe combination of F1 / R1-P1, F2 / R3-P1, and F5 / R5-P2, under the conditions of constant temperature at 40.0℃ and MIRA amplification for 20 minutes, combined with colloidal gold LFD detection, can rapidly and accurately detect EHP in shrimp. The operation is simple, requires no complex equipment, and is suitable for rapid screening of EHP in aquaculture sites.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for detecting Enterocytozoa hepatisci in shrimp using a multi-enzyme isothermal amplification-colloidal gold method, characterized in that... The method includes the following specific steps: Specific primer and probe design: Based on the EHP-specific PTP3 gene, primer pairs and probes are designed according to specific parameters, and the primer concentration is appropriate and the probe labeling and modification meet the detection requirements. Nucleic acid extraction from samples: Specific tissues of shrimp suspected of being infected with EHP were selected, and nucleic acid was extracted using a kit according to standard procedures. The quality of the nucleic acid was then tested. MIRA amplification reaction: Prepare the MIRA reaction system by mixing buffer, primers, enzyme mixture, and template nucleic acid components in the correct proportions, and react under suitable isothermal conditions to complete the efficient amplification of the target gene; Colloidal gold LFD detection: Add the amplification product to the sample well of the colloidal gold test strip, let it stand at room temperature, and judge the test results according to the appearance of the bands in the detection area and the quality control area according to the standard rules.

2. The method for detecting Enterocytozoa hepatica in shrimp according to claim 1, characterized in that, In the specific primer and probe design step, primer pairs and probes are designed using Primer software. The primer length is 25-30 bp, the GC content is 45%-55%, the probe length is 30-35 bp, the 5' end is labeled with a FAM fluorescent group, a THF modification site is inserted in the middle, the 3' end is labeled with a C3spacer quencher group, and the primer concentration is set to 10 μmol / L.

3. The method for detecting Enterocytozoa hepatica in shrimp according to claim 1, characterized in that, In the sample nucleic acid extraction step, nucleic acid was extracted using a kit according to the standard procedure: 200 μL of lysis buffer and 20 μL of proteinase K were added to the tissue, and the mixture was incubated at 56°C for 30 minutes until the tissue was completely digested; then 200 μL of anhydrous ethanol was added, and the mixture was inverted and mixed before being transferred to an adsorption column. The column was centrifuged at 12,000 rpm for 1 minute, and the waste liquid was discarded; 500 μL of low-salt buffer was added, and the column was centrifuged at 12,000 rpm for 1 minute, and the waste liquid was discarded; then 700 μL of ethanol buffer was added, and the column was centrifuged at 12,000 rpm for 1 minute, and the waste liquid was discarded and repeated once; then the empty column was centrifuged at 12,000 rpm for 2 minutes to remove residual washing liquid; 50 μL of enzyme-free water was added to the center of the adsorption column, and the column was allowed to stand at room temperature for 5 minutes, then centrifuged at 12,000 rpm for 2 minutes to collect the eluent.

4. The method for detecting Enterocytozoa hepatica in shrimp using a multi-enzyme isothermal amplification-colloidal gold method according to claim 1, characterized in that, In the sample nucleic acid extraction step, the nucleic acid concentration and purity are determined using a UV spectrophotometer, requiring a concentration of 10-100 ng / μL and an OD260 / OD280 ratio of 1.8-2.

0.

5. The method for detecting Enterocytozoa hepatica in shrimp according to claim 1, characterized in that, In the MIRA amplification reaction step, a 25 μL MIRA reaction system is constructed, including 12.5 μL of 2×MIRA buffer to provide the required pH environment and dNTPs; 1 μL of 10 μmol / L upstream primer for binding to the upstream sequence of the target gene; 1 μL of 10 μmol / L downstream primer for binding to the downstream sequence of the target gene; 0.5 μL of DNA helicase for unwinding the double-stranded DNA of the target gene; 0.5 μL of recombinase for mediating the binding of primers to the single strand of the target gene; 0.5 μL of SSB protein for stabilizing single-stranded DNA; and 0.5 μL of DNA polymerase for extending primers and synthesizing new DNA strands. 2 μL of template nucleic acid, i.e., the nucleic acid of the sample to be tested; 5 μL of enzyme-free water, used to complete the system volume.

6. The method for detecting Enterocytozoa hepatica in shrimp according to claim 1, characterized in that, In the MIRA amplification reaction step, the prepared reaction system is gently mixed and placed in a constant temperature water bath at 25–42°C for 5–20 minutes.

7. The method for detecting Enterocytozoa hepatica in shrimp according to claim 1, characterized in that, In the colloidal gold LFD detection step, the colloidal gold side-flow chromatography test strip consists of a sample pad, a colloidal gold conjugate pad, a nitrocellulose membrane, and an absorbent pad. The colloidal gold conjugate pad is coated with a monoclonal antibody-colloidal gold complex containing an anti-FAM fluorescent group. The detection area on the NC membrane is coated with anti-biotin antibody, and the control area is coated with goat anti-mouse IgG antibody.

8. The method for detecting Enterocytozoa hepatica in shrimp according to claim 1, characterized in that, In the colloidal gold LFD detection step, take 10 μL of MIRA amplification product, drop it onto the sample pad of the test strip, and let it stand at 20-25℃ for 5-8 minutes until the liquid has completely migrated to the absorbent pad.

9. The method for detecting Enterocytozoa hepatica in shrimp according to claim 1, characterized in that, In the colloidal gold LFD detection procedure, a positive result is indicated by clear red bands appearing in both the detection area (T zone) and the control area (C zone), indicating the presence of the EHPPTP3 gene in the sample; a negative result is indicated by a red band appearing only in the control area (C zone), indicating the absence of the EHPPTP3 gene in the sample; an invalid result is indicated by the absence of a red band in the control area (C zone), regardless of whether the detection area (T zone) shows color, indicating that the test strip is invalid and needs to be retested.

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